Heavy metal complexes of trihydroxamic acids

ABSTRACT

THE INVENTION IS CONCERNED WITH HEAVY METAL COMPLEXES OF THE COMPOUNDS OF THE FORMULA   R1-NH-(CH2)5-N(-OH)-CO-(CH2)2-CO-NH-(CH2)5-N(-OH)-CO-   (CH2)2-CO-NH-(CH2)5-N(-OH)-CO-R2   IN WHICH CO-R2 REPRESENTS CARBOXYLIC ACID ACYL AND R1 STANDS FOR A MEMBER SELECTED FROM THE GROUP CONSISTING OF HYDROGEN, CARBOXYLIC ACID ACYL, M-DINITROPHENYL AND, TOGETHER WITH CO-R2 FOR SUCCINYL IN WHICH THE SECOND CARBOXYL IS COMBINED WITH THE N1-ATOM TO FORM A LACTAM, EACH OF SAI CARBOXYLIC ACID ACYL GROUPS BEING A MEMBER SELECTED FROM THE GROUP CONSISTING OF ALKANOYL, ALKENOYL, SUCCINYL, ESTERIFIED SUCCINYL, GLUTARYL, ETHOXYCARBONYL, AMINO-CARBONYL, ETHYLAMINO-CARBONYL, NATURAL A-AMINOCARBOXYLIC ACID ACYL, BENZOYL, SALICYL, P-HYDROXY-BENZOYL, DIHYDROXY-BENZOYL, P-ETHOXY-BENZOYL, P-ETHOXY-ETHOXYBENZOYL, P-ETHOXY-ETHYLENOXY-BENZOYL, NAPHTHOYL, PHTHALOYL, CARBOBENZOXY AND PHENYLACETYL. THE COMPOUNDS OF THE INVENTION ARE USEFUL AS GROWTH PROMOTING SUBSTANCES.

E. GAEUMANN ET AL Jan. 11, 1972 3,634,407

HEAVY METAL COMPLEXES OF TRIHYDROXAMIC ACIDS Original Filed May 18, 1964 8 Sheets-Sheet 1 5 15120 :35 5o 50105 Hi H\ fi FRHCTION NUMBER Jan. 1 1," 1972, E GAEUMANN ET AL HEAVY METAL COMPLEXES OF TRIHYDROXAMIC ACIDS Original Filed May 18, 1964 8 Sheets-Sheet 2 E MZSm FIG. 2

Jan. 11, 1972 GAEUMANN HAL 3,634,407

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Jan. 11, 1972 GAEUMANN ETAL 3,634,407

HEAVY METAL COMPLEXES OF TRIHYDROXAMIC ACIDS Original Filed May 18. 1964 8 Sheets-Sheet E N Q Q 9. Z '2 3 o l l I I l l l L FIG 5 FIG. 4

Jan. 11, 1972 5; GAEUMANN ETAL 7 3,634,407

HEAVY METAL COMPLEXES OF TRIHYDROXAMIC ACIDS Original Filed May 18, 1964 8 Sheets-Sheet 5 OIG 7 I I I I I I I 1' I00 200 300 400 500 600 700 800 900 i000 H00 200 I I I I I I I I T 50 I00 I50 200 250 300 350 400 450 500 E. GAEUMANN ETAL 3,634,407

HEAVY METAL COMPLEXES OF TRIHYDROXAMIC ACIDS Jan. 11, 1972 8 Sheets-Sheet 6 Jan. 11, 1972 E, GAEUMANN ETAL 3,634,407

HEAVY METAL COMPLEXES O-F TRIHYDROXAMIC ACIDS Original Filed May 18, 1964 a SheetS Sheet 7 FIG. IO W FERRIOXAMI N 40003000 2000 I I000 900 a TRANSM SSION FIG H 40003000 2000 I500 'ooo 900 000 7oocm-l oolli ..'1 l 'lll z 00 o R, g so r r""- 1 H hn PM 2 l 1 1 n J \f z W vn U 0-20 .o N 0 FIG. I2 0 Jan. 11, 1 972 3,634,407

s HEAVY METAL COMPLEXES OF TRIHYDROXAMIC ACIDS Original Filed May 18, 1964 v E. GAEUMANN ET AL 8 Sheets-Sheet 8 m W I A11 mmwww FIG. l5

a G W 'u R I310 05;:

United States Patent 3,634,407 HEAVY METAL COMPLEXES OF TRIHYDROXAMIC ACIDS Ernst Gaeumann, deceased, late of Zurich, Switzerland, by Tino Gaeumann, legal representative, and Vladimir Prelog, Zurich, Hans Bickel, Binningen, and Ernst Vischer, Basel, Switzerland, assignors to Ciba Corporation, New York, N.Y.

Division of application Ser. No. 368,424, May 18, 1964,

now Patent No. 3,471,476, which is a continuation-inpart of application Ser. No. 292,443, July 2, 1963, now Patent No. 3,247,197, which is a continuation-in-part of application Ser. No. 144,325, Oct. 11, 1961, which in turn is a continuation-impart of applications Ser. No. 57,834, Sept. 22, 1960, now Patent No. 3,153,621, and Ser. No. 184,870, Apr. 3, 1962. Divided and this application June 19, 1969, Ser. No. 844,712

Claims priority, application Switzerland, Sept. 25, 1959, 78,652/59, 78,653/59; Mar. 18, 1960, 3,063/60, 3,064/ 60; Oct. 11, 1960, 11,395/60; Nov. 23, 1960, 13,147/60; Apr. 7, 1961, 4,075/61; Apr. 26, 1961, 4,885/61; May 18, 1961, 5,831/61; June 29, 1961, 7,598/61; Aug. 10, 1961, 9,409/61; Aug. 11, 1961, 9,451/61; Sept. 14, 1961, 10,685/61; July 6, 1962, 8,185/62 Int. Cl. (307d 41/00 U.S. Cl. 260-239.3 8 Claims ABSTRACT OF THE DISCLOSURE The invention is concerned with heavy metal complexes of the compounds of the formula HO O O HO O This is a divisional application of application Ser. No. 368,424, filed May 18, 1964 now US. Pat. No. 3,471,476, which, in turn is a continuation-in-part of our application Ser. No. 292,443, filed July 2, 1963 now US. Pat. No. 3,247,197, which is itself a continuation in part of our application Ser. No. 144,325, filed Oct. 11, 1961 and now abandoned, which in turn is a continuation-in-part of our application Ser. No. 57,834, filed Sept. 22, 1960 now US. Pat. No. 3,153,621, and of our application Ser. No. 184,870, filed Apr. 3, 1962 and now abandoned.

A large number of biologically and physiologically ac tive substances have already been isolated from materials of biological origin, especially plant material, animal organs and micro-organisms. Nothing, however, was known of the wide-speread occurrence of a certain group of growth-promoting substances.

The present invention is based on the observation that from plant organisms, particularly Actinomycetes, and from extracts thereof growth-promoting substances in ice pure or enriched form can be obtained which are hereinafter referred to as ferrioxamines.

The ferrioxamines are organic compounds containing nitrogen and iron. They are of brownish red color and readily soluble in acids and strongly polar solvents, such as water, dimethyl-formamide, glycol, ethyleneglycolmono-methyl-ether as well as in lower aliphatic alcohols, such as methanol. They are further restrictedly soluble in higher aliphatic alcohols, and in aromatic alcohols and phenols, for example in butanol, benzyl alcohol and phenol. The hydrolysates of the ferrioxamines contain substances of positive reaction to ninhydrin. Chemically, the ferrioxamines are related to a group of antibiotic substances, called"Sideramycins. To the sideramycins belong, inter alia, the iron-containing antibiotics grisein, albomycin, the ferrimycins, the antibiotic 1787 (H. Thrum, Naturwiss. 44, page 561 (1957)) and the substances L.A. 5352 and LA. 5937 (P. Sensi and M. T. Timbal, Antibiotics & Chemotherapy 9, page (1959)).

Crude ferrioxamine which for instance is obtained in the course of fermentation of stremptomycetes is usually a mixture of various components. Ferrioxamine B is the main product of the ferrioxamines formed during the fermentation of S. pilosus Ettlinger et a1. NRRL 2857 (ETH 21748). In addition, other substances are produced which are designated ferrioxamines A, C, D D E and F. By Craigs distribution of the crude product obtained from the culture filtrate (2 to 10 days fermentation at 27 C.) by extraction with a mixture of Hot phenol and chloroform (1 g.:1 cc.), 5 fractions are obtained whose extinction at 425 mm is shown in FIG. 1. From the main fraction III there is obtained by chromatography on the ion exchange resin Dowex 50-WX pure ferrioxamine B hydrochloride as a brown-red powder (FIG. 2, fractions 93-125).

Ferrioxamine B hydrochloride is soluble in water and strongly polar organic solvents. In paperchromatography and multiple distribution it behaves as a uniform substance of similar polarity to ferr'nnycin A and A but differs from these by its considerably greater stability. In a weak acetic acid solution it travels in the course of electrophoresis with only a slightly lower speed than the ferrimycins.

The following properties were found for ferrioxamine B hydrochloride: Microanalysis after 48 hours at 20 C., under 0.001 mm. of pressure: C, 46.17%; H, 7.46%; N, 12.90%, Cl, 5.29%; Fe, 8.54%; P, 0%; S, 0%. Titration: pK* MCS (Helv. 37, 1872 (1954)); 9.74; equivalent weight: 704. Absorption spectrum in water: A 430 m with Infrared spectrum in heavy paraflin oil inter alia bands at 3230, 2900, 1640, 1573, 1461, 1377, 1260, 1225, 1185, 1132, 1028 cm. double bands at 989, 975, 935, 810, 750 cm.- see FIG. 3. Partition coefficient, paperchromatography and paperelectrophoresis: see Table 1, FIG. 4 and FIG. 5.

In the course of hydrolysis with dilute hydrochloric acid the following compounds can, inter alia, be identified: succinic acid, l-amino-S-hydroxylamino-pentane, cadaverine and hydroxylamine. When the hydrolysis is carried out with hydriodic acid, neither l-amino-S-hydroxylamino-pentane nor hydroxylamine is formed. When reacted with 2:4-dinitro-fluorobenzene, ferrioxamine B forms a 2:4-dinitro-phenyl derivative.

From the by-fractions II, IV and V (of FIG. 1) obtained by Craigs distribution of the crude ferrioxamine mixture there can be isolated by chromatography on ion exchange columns further compounds containing iron and having antisideramycin activity (see below). According to their behaviour in paperchromatography (FIG. 4) and on ion exchange columns they are designated ferrioxamine A, C, D D E and F, respectively.

Ferrioxamines A and C isolated from fractions II and IV respectively (cf. FIG. 1) are in their physical-chemical behaviour very similar to ferrioxamine B. In paperchromatography and in multiple distribution A is slightly more polar than B. In the case of C it is the other way round. This finding corresponds to the slightly increased basicity of A as compared with B and its slightly greater electrophoretic migration velocity in weak acetic acid solution, and to the slighter basicity and electric mobility of C as compared with B, respectively (FIG. 5). A and C also show infra-red spectra and solubility properties similar to ferrioxamine B and, like the latter, could till now only be obtained as hydrochlorides in amorphous form.

Ferrioxamine A hydrochloride is a brown-red powder which is readily soluble in water, methanol, alcohol, glacial acetic acid and dimethylformamide. It is insoluble in ether, acetone, ethyl acetate and chloroform. R, in solvent system I: 0.35, in solvent system V: 0.21 (Table 1). Partition coefficient in system VI: 0.111 (Table I). Paper electrophoresis cf. FIG. 5. Microanalysis: C 44.21%, H 7.52%, N 12.63%, Fe 7.95%, Cl, 5.93%. Titration: pK :9.79, equivalent weight: 634. Ultraviolet spectrum in water: x 43o m 1 The infrared spectrum in potassium bromide shows: inter alia bands at 2.92 1 (s.), 3.42 1 (m.), 6.10 1 (s.), 6.32 1 (s.), 6.88 1 (m.), 7.30 1 (w.), 7.92 1 (w.), 8.10 1 (w.), 8.49 1 (w.), 8.98 1 (w.), 9.55 1 (w.), 10.15 1 (w.), 10.67 1 (w.) (see FIG. 11).

Ferrioxamine A gives a positive color reaction with ninhydrin. The iron bound in ferrioxamine A is removed from the complex when subjected to the action of a mineral acid or of strong ailkali. Iron free ferrioxamine A is colorless. It can be transformed back to ferrioxamine A with ferric chloride. It reacts also with the other metal ions with formation of the corresponding metal complexes, for example the greenish colored copper-complex. For further characteristics see Table 1.

Ferrioxamine C hydrochloride shows approximately the same solubilities as A. R, in solvent system I: 0.54, in solvent system V: 0.37 (Table I). Paper electrophoresis see FIG. 5; partition coefficient in system VI: 0.489 (Table I). Microanalysis: C 48.33%, H 7.92%, N 10.20%, Cl 5.15%, Fe 6.82, Titration: pK 8.88; equivalent weight 762. Ultraviolet spectrum in water: max

The infrared spectrum in potassium bromide shows inter alia bands at: 2.92 1 (s.), 3.43 1 (s.), 5.85 1 (m.), 6.10 1 (s.), 6.33 1 (s.), 6.87 1 (s.), 7.30 1 (m.), 7.95 1 (m.), 8.23 1 (w.), 8.52 1 (m.), 9.65 1 w.), 13.23 1 (m.) (see FIG. 12).

Ferrioxamine C gives a positive color reaction with ninhydrin. The iron bound in ferrioxamine C is removed from the complex when subjected to the action of a mineral acid or of strong alkali. Iron free ferrioxamine C is colorless. It can be transformed back to ferrioxamine C with ferric chloride. It reacts also with other metal ions with formation of the corresponding metal complexes, for example the greenish colored copper-complex.

The lipophilic ferrioxamines D D and E isolated by ion exchange chromatography from fraction V (FIG. 1), which in the solvent systems V and VI (Table I) show greater R, values than 0.5 and distribution coefiicients above 1, behave as neutral compounds in electrophoresis and titration (cf. Table 1, FIG. 4 and FIG. 5). Ferrioxamine D which is isolated by ion exchange chromatography as the most rapidly travelling substance from a band appearing uniform (cf. FIG. 8) can be separated by simple distribution between chloroform and water into the more lipophilic ferrioxamine D crystallizing in the form of long red prisms from a mixture of methanol and ether, and into ferrioxamine D which is only formed in very small quantities. Ferrioxamine D is readily soluble in water, methanol, alcohol, glacial acetic acid, methyl Cellosolve and chloroform, sparingly soluble in ether, acetone, ethyl acetate, pyridine and dimethylformamide. It crystallizes from a mixture of methanol and ether in red needles. After being crystallized three times it melts at 194200 C. R, in solvent system I: 0.73, R; in solvent system V: 0.72 (Table I). Partition coefficient in system VI: 1.80 (Table I). Electrophoresis see FIG. 5. Microanalysis: C 49.06%, H 7.50%, N 12.56%, Cl 0%, Fe 8.45%. Titration: no acid or basic functions detectable. Ultraviolet spectrum in water: A 430 m 1,

Infrared spectrum in potassium bromide shows inter alia bands at 2.95 1 (s.), 3.06 1 (s.), 3.25 1 (w.), 3.43 1 (s.), 6.08 1 (5.), 6.35 1 (5.), 6.86 1 (s.), 7.30 1 (m.), 7.94 1 (m),

8.20 1 (w.), 8.49 1 (w.), 8.83 1 (w.), 9.00 1 (w.), 9.65 1 (w.), 10.00 1 (w., 10.31 1 (w.), 10.67 1 (w.), 12.20 1 (w.), 13.30 1 (m.), see FIG. 13. Ferrioxamine D gives no color reaction with ninhydrin. The iron bound in ferrioxamine D is removed from the complex when subjected to the action of a mineral acid or of strong alkali. Iron free ferrioxamine D is colorless. It can be transformed back to ferrioxamine D, with ferric chloride. It reacts also with other metal ions with formation of the corresponding metal complexes, for example the greenish colored copper-complex. Ferrioxamine D Infrared spectrum in potassium bromide shows inter alia bands at: 5.95 1 (s.), 3.43 1 (m), 6.08 1 (s.) 6.36 1 (s.), 6.90 1 (s.), 8.49 1 (w.), 8.87 1 (w.), 9.65 1 (w.),10.05 1(w.),1O.70 1(w.),13.22 1(w.), see FIG. 16. R in solvent system I: 0.64, R in solvent system V: 0.48 (Table I). Paper electrophoresis, see FIG. 5. Ferrioxamine E which has more differentiated infrared spectrum than the other ferrioxamines (see FIG. 14) also differs from them by its poor solubility in water and methanol.

Microanalysis: C, 49.80%, H, 7.37%, N, 12.48%, Cl, 0%, Fe, 8.14%.

Titration: no acid or basic functions detectable.

Ultraviolet spectrum in water: )r 430 m The infrared spectrum in potassium bromide shows inter alia bands at 2.92 1 (s.), 3.02 1 (s), 3.45 1 (s), 5.96 1 (s), 6.15 1 (s), 6.36 1 (s), 6.90 1 (s), 7.10 1 (m), 7.15 1 (m), 7.39 1 (m), 7.82 1 (w), 7.98 1 (m), 8.45 1 (w), 8.54 1 (w), 8.85 1 (w), 9.01 1 (w), 9.20 1 (w), 9.98 1 (m), 10.07 1(w), 10.23 1 (w.), 10.43 1 (W.), lO.7l 1 (w.), 11.78 1 (w.), 13.20 1 (m), 13.66 1(w), (see FIG.14).

Ferrioxamine E gives no color reaction with ninhydrin. The iron bound in ferrioxamine E is removed from the complex when subjected to the action of a mineral acid or of strong alkali. Iron free ferrioxamine E is colorless. It can be transformed back to ferrioxamine E with ferric chloride. It reacts also with other metal ion with formation of the corresponding metal complexes, for example the greenish colored copper-complex. Ferrioxamine F, which according to its behaviour in paperchromatography and in counter-current distribution also belongs to the lipophilic group (D D B) shows however in contrast to D D and E, basic properties and is isolated as hydrochloride (cf. FIG. 4 and FIG. 5 Ferrioxamine F hydrochloride is readily soluble in water, methanol, pyridine, glacial acetic acid, ethanol, dimethylformamide; sparingly soluble in chloroform, insoluble in ethyl acetate, acetone and ether. Microanalysis: C, 50.44%, H, 7.29%, N,

10.53%, Cl, 4.10%, Fe, 5.57%. R; in solvent system V: 0.80 (Table 1). Partition coeflicient in system VI; 3.12 (Table I). Electrophoresis see FIG. 5. Titration: pK 9.75, equivalent weight 695. The infrared absorption spectrum in potassium bromide shows inter alia bands at 2.95 1

cinic acid, citric acid, tartaric acid, mandelic acid, glutamic acid, pantothenic acid, maleic acid, fumaric acid, benzoic acid, cinnamic acid, salicylic acid, para-amino-salicylic acid, Z-phenoxy benzoic acid, 2-acetoxy-benzoic acid, methane eulfonic acid, ethane sulfonic acid, methi- 5 (s.), 3.45;]. (m.), 6.10 t (s.), 6.37 4 (s.), 6.92 7 (m.), 7.4077. onine, tryptophane, lysine or arginine. They are neutral (w.), 7.97 4 (W.), 8.50; (W.), 8.88 (w.), 9.72 (W.), or acid salts. They are prepared by double conversion of 10.10 1 (w.), 10.70;. (w.), 13.75 1. (W.), (see FIG. salts, for example of ferrioxamine sulfate with calcium Ultraviolet absorption in H O: k 430 mg, pantothenate, or by anion exchange on anion exchangers, E170 34 10 for example of ferrioxamine chloride on a strongly basic exchanger, e.g. Amberlite IRA 400 in the sulfate form.

TABLE 2 Analytical values 4 Absorption Substance C H N 01 Fe Molec. weight pK Mcs Ami... E Identified hydrolysis products Ferrimycin A 1 48. 65 7. 09 12. 95 6. 10 4. 56 B l, 106 4. 18;7. 88 228 282 NHa, succinic acid, 1-a1nino5-hydroxyl- (0. 83)

319 28. 2 amino-pentane. fi-aminovalerio acid, 425 22. 6 cadaverine, cryst, substance with km.

227 and 323 m proline and unidentified ninhydrin-positive substances. Grisein A 43. 95 5. 65 12. 97 5. 14 1,034 2 03 Methyluracil, glutamic acid.

8. Albomycin 3 4. 16 b 1, 270-1, 346 Methyluracil, serine, ornithinc Ferrloxamine B 48. 04 7.41 11.21 5. 7. 67 B 704 9. 74 430 39.0 NHa, succinic acid,1-amino-5-hydroxyl- (1.2)

hydrochloride. amino-pentaue, cadaverine, 5-a1'ninovaleric acid, hydroxylamine, no glycine, ornithine or serine. Ferrichrorne 4 44.02 5.90 16. 55 7.35 725 425 39.4 N s.glyCl!1e, ornithine. 2. 89 Ferrichrome A 44. 75 5. 80 11. 18 5. 3 a 1, 100 440 33. 8 Serine, glycine, ornithine. 3. 01 Coprogen 5 50. 96 6. 83 10. 26 6. 61 440 36. 0

1 US. patent application Ser. N 0. 32,294 filed May 27, 1960 by us. a By titration. I F. A. Kuehl al., J. Amer. Chem. Soc. 73, 1770 (1951). b According Fe-, 804- and N Hz-content. 3 G. F. Gause, Brit. Med. J. 1955, 1177. c Found by two different methods. 4 J. B. Neilands, Bact. Rev. 21, 101 (1957). W. Simon, E. Kovats, L. H. Chopard-dit'lean and E. Heilbronner, 5 G. W. Hesseltine et al., J. Amer. Chem. Soc. 74 1362 (1952). Holy. 37, 1872 [1954]. n T Z C aky, Acta Chem. Scand. 2, 450 (1948). Hydroxylarnine-values per atom Fe (found colorimetrlcally aecording to Csaky 6); bracketed values are uncorrected.

The ferrioxamines promote the growth of a large number of organisms. For instance, they have such an effect on Bacillus sublillis, Micrococcus pyogenes var. aureus, Saccharomyces ceravisiae, Ustilago phaerogena and Chlamydomonas eugametos. In Table 3 there are summarized as examples the results of tests with Ustz'lago sphaerogena (smut fungus) and with Chlamydomonas sugametos (Chlorophyceae) for which an enriched preparation of ferrioxamine B was used.

are shown by way of comparison.

TABLE I Countercurrent Paper distribu- Extinction Titration chromatography tion at 430 mg Paper electro- Equivalent Ferrioxamine phoresis e RrI RrV K VI E 1% 1 cm. DKM'CS Weight 13. 6 0.35 0. 21 0. 11 37 9.89 634 13.0 0.44 0.29 0.23 39 9. 74 704 12. 3 0.54 0. 37 0. 49 39 8. 88 762 3. 9 0. 73 0.72 1. 80 44 (Neutral) 3. 9 0.64 0. 48 (Neutral) 3. 9 0. 68 0.59 1. 59 42 (Neutral) 12.5 0. 80 3.12 34 9.

RyIZ Pei-value in system lzn-butanol-glacial acetic acid-water (4: 1:5)

RrV: Rr-value in system Vztertiary butanol-Water-saturated aqueous sodium chloride solution0.1 N H01 (:25:2511), paper impregnated with acetone-water-saturated aqueous sodium chloride solution (6:3:1).

K VIzpartition coeI-Iicient in system VIzn-butanol-benzyl alcohol-water-saturated aqueous sodium chloride solution-0.1 N H01 (200:100z300z60z3). Distribution of 10 mg. over 34 stages each of 3 cm. organic and 3 cm. aqueous phase at 2325 0. Evaluation by measuring the extinction at 425 mu (2 cm. of the fractions diluted with alcohol to 10 cm.).

"W. Simon et al., Helv. 37, 1872 (1954).

TABLE 3 Relative growth compared with an untreated control specimen Ferrioxamine B addition (rig/cc.)

Ustz'lago sphaerogena (24 hours culture), percent 100 540 405 247 158 111 Chlamydomonas eugametos (4 days culture), percent 100 280 271 136 98 98 Other organismsfor example representatives of the genus Arthrobacter, such as Arthrobacter terregens and Arthrobacter flavescens-can develop at all only in the presence of ferrioxamine; that is to say that in these cases the ferrioxamines have a vitamin-character similar to that observed for the growth factors ferrichrome, terregens factor and coprogen (Bact. Rev. 21, page 101 [1957] It is another biological property of the ferrioxamines that they are capable of counteracting competitively the antibacterial action of antibiotics belonging to the group of the sideramycins towards Gram-positive organisms.

The antisideramycin action of the ferrioxamine is shown by the antibiotics albomycin, grisein, A 1787, ferrimycin and A 22765, all of which also display a crossresistance with grisein. It is surprising that the ferrioxamines counteract the action of the grisein-like sideramycins towards Gram-positive bacteria but not towards Gram-negative bacteria.

The antagonism between the ferrioxamine and the sideramycins, which is called antisideramycin activity, can be observed both in vitro and in vivo. The antagonising action of ferrioxamine B on the antibacterial activity of various antibiotics is shown in the following table. The test organisms used were Escherichia coli, Bacillus sabtilis and Staphylococcus aureus. The antibiotics were tested in the modified test according to Bonifas towards ferrioxamine B (1 mg. per cc.), which is described below.

Competitive Competitive d d Nil.

Streptothricim. Viomycin Penicillin Nil Nil 1 signifies that the antibiotic so marked is practically inactive towards this test organism.

7 Strain S. aurcofacicns Duggar A 22765.

Further antibiotics not affected by ferrioxamines, not shown in the above table are: Acetomycin, the actinomycins C, X, I and Z, angolamycin, carbomycin, chartreucin, chlorotetracycline, cycloserin, the cinerubines, desertomycin, erythromycin, exfoliatin, granaticin, holomycin, leucomycin, megacidin, methymycin, narbomycin, novobiocin, oleandomycin, oxytetracycline, picromycin, rhodomycin, the spiramycines, streptogramin, the tetriomycines and thiolutin.

The antisideramycin activity in vitro of the ferrioxamines facilitates a qualitative indentification and quantitative determination of the ferrioxamines, since the test developed by Bonifas (V. Bonifas, Experientia 8, page 234 [1952]) specifically for the determination of synergistically active substances can be suitably applied. For this purpose plates, e.g. patri dishes containing a layer of a suitable agar medium are inoculated with Bacillus subtilis Cohn amend. Praxmowski or StaplzylOcoccus aureus Rosenbac are prepared. Strips of filter paper, for instance of Whatmann paper No. 1 mm. wide) impregnated with a solution of a sideramycin antibiotic, for example IO'y/cc. ferrimycin in methanol are placed on these agar layers. At right angles other strips, likewise of 5 mm. width, saturated with the solution whose content of ferrioxamine is to be tested are placed on the sideramycin containing strips. After incubation for 9-15 hours at 36 C. the influence of the ferrioxamines on the antibiotic activity of ferrimycin is easy to recognise. In the inhibition aureole which forms from the strips saturated with the antibiotic there is formed, at the crossing of the two strips, a wedge-shaped constriction, the form and dimensions of which under standard conditions are used for the 8 quantitative determination of the ferrioxamine concerned (see FIG. 10)

If a solution contains both ferrimycin and ferrioxamine, it must be heated to 60 C. for 30 minutes at neutral pH. By this procedure the antibiotic activity of the ferrimycin is destroyed, while the ferrioxamine remains unaffected and can consequently be determined quantitatively.

In this antisideramycin test ferrioxamine B is the most eifective of all the ferrioxamines. With Staphylocoocus aul'eus as test organisms the other ferrioxamines show the following activities in relation to B: A 51%, C 16%, D 8%, D 3%, 134% andF 30%.

The aforementioned substances ferrichrome, terregens factor and coprogen resemble the ferrioxamines in their vitamin-character with respect to Arthrobacter terregens and Arthrobacter flavescens. On the other hand the aforementioned typical growth-promoting effect of the ferrioxamines on Bacillus subtilis, Micrococcus pyogens, Saccharomyces cerovisiae, Ustilago sphaerogena and Chlamydomolzas eugametos has not been observed with the three substances ferrichrome, terregens factor and coprogen. Furthermore nothing is known about an antagonistic effect of these substances on the antibacterial activity of the sideramycin antibiotics, which is typical for the ferrioxamines.

In paperchromatography ferrichrome differs distinctly in direct comparison in the system V (cf. Table I) from the ferrioxamines B-F, but shows in this system a similar R -value to ferrioxamine A. On the other hand, as neutral substance it is easily distinguished from the strongly basic ferrioxamine A in paper-electrophoresis in 0.33 'N-acetic acid. The terregens factor contains only traces of iron and is consequently different from all the ferrioxamines. Coprogen can be easily distignuished from the ferrioxamines A, B, D and E by means of its analytical data. The ratio percent C/ percent N is in the case of coprogen 4.97, in the case of the ferrioxamines 3.5-4.3. In the case of ferrioxamines C and F this difference is not so pronounced (percent C/percent N=4.744.78). Ferrioxamines C and F are strong bases which are isolated as hydrochlorides, whereas coprogen, according to the present data, is a neutral substance (J ourn. Amer. Chem. Soc. 74, 1362 1952) The ferrioxamines, their derivatives and fission products and the salts of these compounds are obtained by isolating the new growth-promoting substances from plant organisms or extracts thereof by methods known per se taking the above chemical and physical data into consideration and using the antisideramycin test and, if desired, the salts, derivatives or fission products of the new compounds are prepared.

Starting materials suitable for the preparation of the ferrioxamines aref or example: Organs of higher plants such as dicotyledoneac, e.g. Solanaceae, for instance Solanum lycopersicum L. or Umbelliferae, for instance Daucus carota L., and Monocotyledoneae, e.g. Commelinaceae for instance Rhoeo discolor (L. Hr.)Hance, cultures of algae, for example of Chlamydomonas eugametos, or above all cultures of microorganisms, for example of representatives of the genus Streptomyces, of bacteria for example of B. subtilis or of yeasts, for example of Saccharomyces cerevisiae. The antisideramycin activity can be observed by way of the aforementioned test either in the crude extract or in the culture filtrate.

An especially preferred source are cultures of strains of Streptomycetes which according to the characteristics proposed by Ettlinger et al. (Arch. Mikrobiol, 3, page 326 [1958]) belong to the following species: Streptomyces griseoflavus (Krainsky) Waksman et Henrici, Streptomyces lavendulae (Waksman et Curtis) Waksman et Hemrici, Streptomyces galilaeus Ettlinger et al., Streptomyces pilosas Ettlinger et al., Streptomyces polychromogenes Hagemann, Penasse et Teillon, Streptomyces viridochromogenes (Krainsky) Waksman et Henrici, Streptomyces aureofacicns Duggar, Streptomyces olivaceus (Waksman) Waksman et Henrici, Streptomyces griseus (Krainsky) Waksman et Henrici, Streptomyces glaucescens Gause et al.

The following table lists the specific features characterizing the Streptomycetes strains capable of producing ferrioxamines.

vent, such as an alcohol, for example aqueous methanol.

In a similar manner also bacteria, for example B. subtilis, can be grown and the culture filtrates used as source for the isolation of the ferrioxamines.

TABLE Morphology of Color of air Melanoid Characteristics species spores mycelium Morphology of air mycelium pigment S. giseqflcvus (Krainsky) Waksman Spore: with short Ash grey Spore chains with open, regular spirals, often over 6 coils Absent.

enrici. Spl es. S. pilosus Ettlinger et a1 Spores with fine ..do "do Present.

brittle hairs. S. viridochromogenes (Krainsky) Spores with short Pale blue -.d0 Do.

Waksman et Henrici. spikes.

a iliv ce us (Waksman) Waksman Smooth Ash grey Spore chains monopedially branched, straight or wavy Absent.

enric S. aureofaciens Duggar do do Spore chains monopcdially branched, with irregular, open Do.

S. galz'laeus Ettlinger et al do ..do.

S. lavendulae (Waksman ct Curtis) do Pale carmine to Waksman et Henrici. cinnamon brown. P U hTOMOQEnBS Hagemann et al ,do Pal e carminc to cinnamon. S. ariseus Waksman et Henrici do Yellowish to greenish grey.

spirals.

Spore chains mouopedially branched, long straight main Present.

axis, open regular spirals, generally more than 6 00115.

Spore chains with open irregular spirals at the ends of long D0.

straight pieces.

Spore chains straight or wavy Absent.

Spore chains Wavy, sympedially branched, bunches Do.

without spirals.

Substantial amounts of the ferrioxamines are advantageously prepared from cultures of the aforementioned micro-organisms. Particularly good results have been obtained in this connection with the aforementioned streptomyces strains which are easy to grow on a large scale. The present invention is however, not restricted to the use of representatives of the aforementioned species but it includes also the use of strains of other species capable of producing the ferrioxamines and more especially of variants of all these organisms such as are obtained, for example, by selection or mutation, more especially with irradiation with ultra-violet or X-rays or under the action of nitrogen mustard oils.

To prepare a substantial amount of the ferrioxamines for example, a strain possessing the properties of the aforementioned Streptomycetes is grown under aerobic conditions, for example in an aqueous nutrient solution containing carbohydrates, nitrogenous compounds and inorganic salts until the solution displays a substantial ferrioxamine action, whereupon the ferrioxamines are isolated. Alternatively, plants can be grown such as Chlorophyceae, or bacteria such, for example, as B. subtilis, and from these the ferrioxamines are isolated in pure or enriched form. Assimilable carbohydrates suitable for growing the aforementioned microorganisms are for example glucose, saccharose, lactose, mannitol, starches or glycerol. Suitable nitrogenous nutrients and if desired growthpromoting substances are: amino acids, peptides and proteins and breakdown products thereof such as peptone or tryptone, also meat extracts, water-soluble constituents of cereal grains, such as maise and wheat, of distillation residues of the manufacture of alcohol, of yeasts, seeds, more especially of the rape and soybean plants, cotton seeds and the like, also ammonium salts and nitrates. Inorganic salts present in the nutrient solution may be, for example, chlorides, carbonates, sulfates of alkali metals, alkaline earth metals, magnesium, iron, zinc or manganese.

The microorganisms are grown under aerobic conditions, for example in a static surface culture or preferably submerged with shaking or stirring with air or oxygen in shaking flasks or in the known fermenters. If Streptomyces strains are used the cultivation temperatures range from 18 to 40 C. Under these conditions the nutrient solution develops a substantial ferrioxamine activity in general Within 2 to days. 0.1% of ferric chloride is then added to the culture and the mycelium is separated from the culture filtrate, whereupon the bulk of the ferrioxamines is found in the culture filtrate. Substantial amounts of ferrioxamines however still remain adsorbed on the mycelium so that the latter is advantageously thoroughly washed, for instance with Water and/or an aqueous organic sol- The ferrioxamines can be isolated from the aforementioned materials, more especially from the culture filtrates of fungus or bacterium cultures, by as such known methods for example by one of the methods mentioned below or by a combination of two or more of such methods:

(1) An adsorbent can be used, for example an active carbon such as Norit, an activated earth, such as Frankonit, fullers earth or fioridine or an adsorber resin such as Asmit. The adsorbates are advantageously eluted with a mixture of water with a water-miscible organic solvent, for example with a mixture of water+methanol, water-{- pyridine, diluent acetic acid-l-methanol or a mixture of water+mcthanol+glacial acetic acid-l-butanol. In eluting a Frankonit or Norit adsorbate particularly good results have been achieved with a mixture of 4 parts by volume of water and 1 part by volume of pyridine.

(2) According to another method of isolation the ferrioxamines are adsorbed on a cation exchanger and for this purpose a resin containing acid groups, such as Dowex-SO, is especially suitable. This resin can be used either in the acid form or in the sodium form, though mixtures of these two forms have proved particularly useful. The elution is advantageously carried out with an acid agent, for example with methanolic hydrochloric acid or an acidic buffer solution.

(3) Furthermore, the ferrioxamines can be extracted from an aqueous solution thereof by means of an organicsolvent. Higher organic alcohols, for example benzyl alcohol or isopropanol, have proved particularly advantageously for this extraction process. It is of advantage in this connection to add to the aqueous phase an inorganic salt, for example ammonium sulfate or sodium chloride. From the resulting organic extracts the ferrioxamines can be obtained in an enriched form either by evaporating the solvent or by precipitating the product with a suitable organic solvent, for example ether, petroleum ether of ethyl acetate.

(4) The ferrioxamines can also be enriched by treating a concentrated aqueous or alcoholic-aqueous solution of the salt with an excess of an organic water-miscible solvent such as acetone, dioxane or the like, whereby the salts are precipitated in solid form.

(5) Another method of enriching the ferrioxamines consists in distributing them between an aqueous solution and a solution of phenol in chloroform, both the pH- value of the aqueous solution and the phenol content of the chloroform solution being varied. Taking as the coefficient of distribution of the ferrioxamines the ratio of their concentration in the organic phase to their concentration in the aqueous phase, it will be realized that the coefiicient of distribution rises as the phenol content of the organic phase is increased, and is reduced as the pH of the aqueous phase is lowered. Since it is thus possible to establish any desired coefiicient of distribution of the ferrioxamines in this system, a combination of a few distribution operations enables a large portion of inactive impurities to be removed.

(6) Another method of enriching the ferrioxamines is chromatography, such as adsorption chromatography on various materials, for example on Norit, alumina, magnesium silicates, silica gel or calcium sulfate, as well as partition chromatography using cellulose, starches, silica gel, Colit or the like as carrier substances, or chromatography on ion-exchanger resins such as Dowex-SO, Amberlite IRC-SO and the like.

(7) Furthermore, the ferrioxamines can be enriched by counter-current distribution according to Craig between two immiscible solvent phases. For this purpose the following solvent systems have proved partciularly advantageous:

(a) Benzyl alcohol-aqueous ammonium sulfate solution of 20% strength.

(b) 100 parts by volume of n-butanol200 parts by volume of benzyl alcohol-6 parts by volume of N- hydrochloric acid-300 parts by volume of water60 parts by volume of aqueous sodium chloride solution saturated at 19 C.

(8) Finally, the purification, enrichment and separation of ferrioxamine preparations can be performed by preparative electrophoresis on a column of carrier material. This process is advantageously carried out as a high-voltage electrophoresis at 500-4000 volts. A further improvement can be obtained by carrying out the electrophoresis according to the so-called counter-current principle, in which the basic ferrioxamines A, B, C and F, which are present as cations are locally anchored on the carrier column by accurately compensating their movement produced by the electric field with a current of electrolyte flowing in the opposite direction. In this way it is ensured that substances having a different electric mobility leave the carrier column at the ends of the two electrodes.

The individual ferrioxamines are obtained as pure and uniform substances in the form of amorphous powders or as crystals. For their preparation the following methods have proved to be useful: lyophilization of an aqueous or alcoholic solution. precipitation from an aqueous, alcoholic or phenolic solution with lipophilic organic solvents which are miscible with the solvent containing the ferrioxamine in question. Particularly suitable for this precipitation are lower alkyl ketones, such as acetone, methylethyl ketone, others, such as diethyl ether, diisobutylether, and hydrocarbon such as pentane, hexane, petroleum ether.

crystallization from suitable solvent mixtures, such as alcohol-ether mixture, like methanol-diethylether, or mixtures or water and organic solvents at least partially miscible with water, like water-acetone, water-glacial acetic acid and so on.

The ferrioxamines as well as their derivatives and their salts can be used for promoting the growth of various organisms, for which purpose they are used as such or in the form of special preparations containing the aforementioned compounds in admixture with a suitable vehicle.

They also possess marked anti-anaemic properties, which were demonstrated in the case of ferrioxamine B as follows in experimental animals:

(1 ANTI-ANAEMIC EFFECTS In the normal rabbit, 10 mg./kg. s.c. daily for 5 days, corresponding to approximately 0.8 mg./kg. Fe produced no significant alterations in the haemoglobin level, in the erythrocyte and reticulocyte counts, or in the haematocrit. On the other hand, from the 3rd day onwards, a certain increase in the sedimentation rate as measured according to Westergren 'Was observed, the figure rising from 20 mm. in 24 hours to approx. 30 mm., then on the 5th day to 40 mm., reverting to normal again from the 6th day onwards. This increase in the sedimentation rate was accompanied by a rise in the total serum proteins as measured electrophoretically, this rise affecting particularly the a and 'y globulins; the total serum protein levels subsequently returned to normal parallel with the normalisation of the sedimentation rate.

(a) Haemorrhagic anaemia By consrast, in a group of 5 rabbits following haemorrhage involving a loss of blood equivalent to 2% of their body-weight, a considerable decrease in heamoglobin (down to approx. 7 g./ ml.) was noted on the next day, followed by relatively slow restitution and virtual normalisation after 24 days. Parallel with this a crisis affecting the reticulocytes was observed, which reached its maximum of 6% around the 9th day and was followed by more or less complete normalisation after 24 days. Under the same experimental conditions, a similar group of 5 rabbits which had received ferrioxamine B in daily doses of 10 mg./kg. s.c. for 24 days, starting from the day after the haemorrhage, showed a much more rapid rise in haemoglobin, the level of which became roughly normal again by the 18th day; in the animals thus treated, the crisis involving the reticulocytes also subsided on the 3rd day. The results obtained in these experiments are listed in Table 6.

TABLE 6 Haemoglobin Retieulocytes Ferrioxa- Ferrioxamine B, 10 mine B, 10 Days Controls ing/kg. s.c. Controls rug/kg. so.

0 haemorrhage 12 12 22 22 7. 2 8. 0 37 37 8. 2 8. 8 44 48 8. 7 9. 1 68 42 8. 7 9. 0 62 37 9. l 10. 0 56 20 9. 3 10. 3 43 25 10. 6 12 34 25 10. 8 12. 5 30 23 12. 2 12. 8 27 21 2% of the animal's body-weight.

In another series of tests, a more severe haemorrhage was produced by first removing blood equivalent to 2% of the animals Weight and then, 3 days later, removing the equivalent of a further 1%. Under these conditions, a group of 5 rabbits showed a fall in haemoglobin to 7.5-8 g./ 100 ml., which reached its maximum 1 day after the second haemorrhage, persisted until about the 9th day, and reverted roughly to normal on the 27th day. At the same time, a reticulocyte crises was noted, which gradually reached its maximum by the 9th day (reticulocytes 6.9%) and then slowly subsided until at the end of the test the reticulocyte count had reverted to normal. A parallel group of 5 comparable rabbits, having received ferrioxamine B in daily doses of 10 mg./kg. s.c. for 27 days, showed a much less pronounced fall in haemoglobin and a much more rapid recovery. At the same time, the reticulocyte crisis attained a maximum of barely 4% between the 3rd and 6th day, normalisation supervening towards the 18th day. These results are listed in Table 7.

Reticuloeytes, percent fying the haemoglobin level, normalising the erythrocyte index, and attenuating the reticulocyte crisis.

(c) Bartonella muris anaemia in rats F ern'oxa- Ferrixa Days Controls 32513 1 Controls fig $3 5 In adult rats suifering from latent infection with Bartonella murls, splenectomy-performed according to the haemrrhagem" method of J. Marmorston-Gottesman and D. Perla (I. a lirifiir'ni' eillil 814 913 413 319 exp. Med. 52, 121, 131, 1930) and W. Weinmann (J. 4 5:3 3-8 2:3 2:2 Infect. Dis. 63, 1, 1938)led to acute anaemia which 8.5 9.5 6.9 3.1 10 often proved fatal. This anaemia was characterised by a 36 2 2:: 3:2 considerable diminution in the erythrocyte count, a fall 10. 7 11 4. 5 2.1 in the haemoglobin level, and an intense reticulocyte crisis '3 g of approx. 10%, these manifestations attaining their maxi- 12.2 12.5 2.5 2 mum degree roughly 6 days after splenectomy. Similar 12% of the animars body weight 15 groups of 10 rats which had received ferrioxamine B in 2 1% of the animals body-weight.

daily doses of 1 0 mg./kg. s.c. or p.o. showed a decidedly smaller decrease in the erythrocyte count and in the haemoglobin level as well as a far less severe reticulocyte crisis, which for example was even suppressed after 10 rug/kg. p.o. The results thus obtained are summarized in Table 9, showing that ferrioxamine B is also capable of exerting its specific anti-anaemic properties in this type of experimental anaemia.

TABLE 9 Erythoeytes, millions/ml, Haemoglobin, g./100 ml. Retieuloeytes Ferrioxamine Ferrioxamine Ferrioxamine B, 10 ing/kg, B, 10 ing/kg, B, 10 mgJkg,

Days Controls S.c P.o. Controls S.o. P.o. Controls S.o, P.o.

0 spenectomy 6- 69 7. 18 7. 52 13.8 13.2 14. 7 26 28 21 3- 6. 76 7. 21 6.83 12.8 12.7 13. 2 44 32 28 6 5. 85 7- 00 6. 56 12. 3 12. 4 12. 7 102 42 31 9 6. 78 7.05 6.71 11. 2 12. 7 12.6 88 46 31 12 6. 06 6.87 7.10 11. 7 12.6 13. 4. 68 67 29 (b) Iron-deficiency anaemia in rats A group of 10 to 15 young rats weighing approx. 40 g. and receiving a diet consisting exclusively of cows milk and semolina, according to the method of D. L. Drabkin and H. K. Miller (J. Biol. Chem. 93, 399, 1931) and E. Rothlin and E. Undritz (Helv. med. Acta, Series A, 460, 1946), developedin addition to slightly retarded growth an iron-deficiency anaemia with a fall in haemoglobin to approx. 7 g./ 100 ml., accompanied by no significant change in the erythrocyte count but by a decrease in the haemoglobin index reaching its maximum after 30-60 days. The reticulocyte crisis attained its maximum after 60 days. Parallel groups of animals which received ferrioxamine B in daily doses of 10 mg./ kg. s.c. or p.o. starting from the 60th day showed a considerable rise in haemoglobin, the level of which reverted almost to normal after 70-80 days; there was little effect on the erythrocyte count, but the haemoglobin index approached normal figures, aand the reticulocyte crisis was less marked than in the controls. The results obtained in these experiments are listed in Table 8.

(2) Other pharmacological properties Ferrioxamine B has a meager spectrum of pharmacological properties: given in doeses of up to 1 00 mg./kg. i.v., it causes no fall in arterial blood pressure and has no influence on respiration in rabbits anaesthetized with urethane (1.4 g./kg. s.c.). On isolated organs, such as the rabbit or guinea-pig intestine, the seminal vesicle of the guinea-pig, and the vessels of the rabbit hindquarters, it is inactive in concentrations of up to IOO' /mL, at which concentrations it also has no specific anticholinergic, musculotropic, adrenolytic, or histaminolytic efiects on the appropriate isolated organs. As a growth factor, ferrioxamine B promoted the proliferation of various micro-organisms, such as B. subtilis, Staphylococcus aureus, Candida vulgaris, Ustilago sphaerogena, and Chlamydomonas eugametos (H. Bickel, E. O'alumann, W. Keller-Schierlein, V. Prelog, E. Vischer, A. Wettstein, and H. Z'aihner: Experientia 16, 129, 1960), in concentrations of O.1'y/rnl. and above. In concentrations ranging up to 10071111. it has no bacteriostatic effect in vitro on any of a Whole series of gram-positive and gram-negative bac- TABLE 8 Days Haemoglobin, g./100 mi. Erythrocytes, IIlilliOIlS/CILIDII'I. Index y-Hb/erythrocyte Retiouloeytes, percent Perrioxamine B, 10 Perrioxamine B, 10 Perrioxamine B, 10 Perrioxamine B, 10

Controls mg./kg. daily p.o. Controls ing/kg. daily p.o. Controls ing/kg. daily p.o. Cont ols mgjkg, daily p.o.

These results shown that, in daily doses of 10 mg./kg. s.c. and p.o., ferrioxamine B was able to exert a specific teria, nor did it display any activity against various fungi in concentrations up to the very high level of 1'001,000

influence on this form of iron-deficiency anaemia, recti- 7 'y/mL; it also showed no chemotherapeutic eifects in doses 15 of up to 500 mg./kg. s.c. administered times in 30 hours, affording no protection to groups of mice suffering from a lethal Staphylococcus aureus or Streptococcus haemolyticus infection.

(3) TOXICOLOGY The toxicity of ferrioxamine B is very low, the LD (amount required to kill 50% of the animals) for a single dose being 950 rug/kg. i.v. and 2,500 mg./kg. p.o. in mice and 500 mg./kg. s.c. in rats. In mice and rats, sublethal doses provoke signs of intoxication in the form of ataxia and non-specific paralysis interrupted by mild convulsions.

In sub-acute toxicity tests, ferrioxamine B was administered to groups of 12 rats for 28 days in daily doses of 1,000 mg./kg. s.c. None of the animals died during the tests. The weight of the animals thus treated increased from 118 to 222 g. and that of the controls from 113 to 214 g. hence, the rate of growth remained perfectly normal. At the same time, a regular check was kept on the red and white blood counts: no pathological changes in the blood such as might have been ascribed to the repeated administration of ferrioxamine B were found. At the end of the experiment, examination of the main organs, such as the heart, lungs, stomach, intestine, liver, kidneys, spleen, thymus, lymph nodes, thyroid, and testicles, showed that there had been no alteration in weight and that no pathological lesions attributable to repeated treatment with ferrioxamine B had occurred.

The acute local tolerability of ferrioxamine B was good; not until a concentration of 30% was reached did slight transient hyperanaemia develop when the product was instilled into the conjunctival sac of rabbits.

(4) SUMMARY AND CONCLUSION Ferriox'amine B displays specific anti-anaemic properties and low toxicity in experimental animals; it may therefore be considered for medicamentous use in the treatment of anaemia in daily parenteral doses of 100 mg. or fractions of the latter.

Suitable vehicles for preparations are substances that do not react with the ferrioxamines such, for example, as gelatine, lactose, starches, magnesium stearate, talc, vegetable oils, benzyl alcohols, gums, polyalkylene, white petroleum jelly or cholesterol. Such preparations may be in solid form, for example powders, or, in liquid form, solutions, suspensions or emulsions. They may be sterilized and/or may contain additives such as preserving, stabilizing, wetting agents or emulsifiers. They may further contain other therapeutically useful substances.

FIG. 1 shows a counter current distribution of crude ferrioxamine in the system n-butanol-benzyl alcoholwater-saturated aqueous sodium chloride solution-N-hydrochloric acid (100:200z300260z6) over 80 stages each of 100 cc. of organic phase and 100 cc. of aqueous phase. Extinction at 425 m Antisideromycin-activity in mm. (modified Bonifas-test).

FIG. 2 shows a chromatogram of fraction III of FIG. 1 on Dowex 50-WX using an ammonium acetate, buffer as eluting agent. Extinction at 425 III/1..

FIG. 3 shows the infra-red spectrum of ferrioxamine B in heavy paraffinic oil.

FIG. 4 shows a paperchromatogram of the ferrioxamines in the system V: tertiary butanol-water-saturated aqueous sodium chloride solution-0.1 N HCl (50:25: 25:1), paper impregnated with acetone-water-saturated aqueous sodium chloride solution (6:3 1).

FIG. 5 shows paperelectrophoresis of the ferrioxamines, cm. path in 0.33 N acetic acid after 4 hours at 220 v. In comparison fructose travels 3.9 cm.

FIG. 6 shows a chromatogram of fraction II of FIG. 1 on Dowex 50-WX using an ammonium acetate buffer as eluting agent. Extinction at 425 m FIG. 7 shows a chromatogram of fraction IV of FIG. 1 on Dowex 50-WX using an ammonium acetate buffer as eluting agent. Extinction at 425 m FIG. 8 shows a chromatogram of fractions 25 obtained by chromatography of fraction V of FIG. 1 on Dowex 50-WX using ammonium acetate buffer as eluting agent in both instances. Extinction at 425 m FIG. 9 shows a chromatogram of fractions 48-55 obtained by chromatography of fraction V of FIG. 1 on Dowex 50-WX using ammonium acetate buffer as eluting agent in both cases. Extinction at 425 m FIG. 10 shows the antagonism between ferrioxamines and ferrimycin in the modified Bonifas-test. The hatched area indicates inhibition of the growth of the test organism.

FIG. 11 shows the infra-red spectrum of ferrioxamine A in potassium bromide.

FIG. 12 shows the infra-red spectrum of ferrioxamine C in potassium bromide.

FIG. 13 shows the infra-red spectrum of ferrioxamine D in potassium bromide.

FIG. 14 shows the infra-red spectrum of ferrioxamine E in potassium bromide.

FIG. 15 shows the infra-red spectrum of ferrioxamine F in potassium bromide.

FIG. 16 shows the infra-red spectrum of ferrioxamine D in potassium bromide.

As already mentioned, from the ferrioxamines the iron can be eliminated, for instance by treating the red-colored solution containing a ferrioxamine, with a mineral acid or a strong alkali. There is obtained a colorless solution containing a desferri-ferrioxamine. By treatment of such colorless solution with ferric chloride the typical color of the ferrioxamines returns.

Now it was found that the desferri-ferrioxamines are 7,18,29 trihydroxy-8,1l,19,22,30-pentaoxo-l,7,12,18,23, 29-hexaaZa-triacontanes. Desferri-ferrioxamine B is the 30-methyl derivative, desferri-ferrioxamine C is the 30-(6- carboxyethyl)-derivative, desferri-ferrioxamine D the 1- acetyl-30-methyl-derivative of 7,l8,29-trihydroxy-8,l1,19, 22,30 pentaoxo-l,7,l2,18,23,29-hexaaza-triacontane and desferri-ferrioxamine E is the 33-membered, cyclic compound in which the carboxyl group at the end of 30-(5- carboxyethyl) 7,18,29 trihydroxy 8,11,19,22,30-pentaoxo-1,7,12,18,23,29-hexaaza-triacetane is combined with the N -atom.

The present invention concerns trihydroxamic acids of the Formula I (1) 1n which R stands for hydrogen, a carboxylic acid acyl radical or an unsubstituted or substituted hydrocarbon radical, CO-R represents an acyl radical or R and CO'R together stand for the radical of a dicarboxylic acid, especially succinic acid, or which the second carboxyl group is combined with the N -atom, the O-acyl derivatives, the salts and metal complexes of said compounds.

An acyl radical R or COR is for example an aliphatic acyl radical, preferably an alkanoyl or alkenoyl radical, e.g. the formyl, acetyl, propionyl, butyryl, valeryl, stearyl or oleyl radical, or a substituted alkanoyl radical, for example a free or esterified e.g. lower alkanoyl-esterified succinyl or glutaryl radical, are ethoxy-carbonyl or amino-carbonyl radical or an amino acid radical preferably one of a natural a-amino acid, e.g. the glycyl, alanyl, valyl or leucyl radical, also an aroyl or aralkanoyl radical, for example an unsubstituted or substituted benzoyl radical, e.g. the salicyl, p-hydroxy-benzoyl, dihydroxybenzoyl, p-aminosalicyl, p-methoxy-benzoyl, p-ethoxy- 17 benzoyl, p-ethoxy-ethoxy-benzoyl, p-ethoxypolyethyleneoxy-benzoyl radical, a naphthoyl, a free or esterified phthaloyl, a carbobenzoxy or phenyl-acetyl radical. When R stands for a hydrocarbon radical then it is preferably an aryl radical especially the m-dinitro-phenyl radical.

The trihydroxamic acids of the Formula 'I are, if R is not an acyl radical, bases, which form salts with acids. For preparing such salts there come into consideration preferably therapeutically acceptable acids, either inorganic acids, for example hydrohalic acids, e.g. hydrochloric or hydrobromic acid, also perchloric, nitric or thiocyanic acid, sulfuric or phosphoric acids, or organic acids, such for example as formic, acetic, propionic, glycellic, lactic, pyruvic, oxalic, malonic, succinic, maleic, fumaric, malic, tartaric, citric, asorbic, hydroxymaleic or dihydroxymaleic acid, benzoic, phenylacetic, 4-aminobenzoic, 4-hydroxy-benzoic, anthranilic, cinnamic, mandelic, salicylic, 4-amino-salicylic, 2-phenoxy-benzoic, 2- acetoxy-benzoic acid, methane sulfonic, ethane sulfonic, hydroxyethane sulfonic, benzene sulfonic, p-toluene sulfonic or sulfanilic acid, methionine, tryptophane, lysine or arginine.

The O-unsubstituted compounds are also of acidic character and therefore form salts with bases. The latter are preferably those of therapeutically acceptable alkali or alkaline earth metals, e.g. of sodium, potassium or calcium or of organic bases, for example aliphatic amines. The O- unsubstituted compounds also form metal complexes. Preferred metals for the latter are such as are physiologically tolerable, preferably iron, cobalt or magnesium, also copper or antimony.

Owing to their capacity to form very stable complexes with metals, e.g. the above shown metals, the compounds of Formula I have valuable pharmacological properties. For example, they inhibit the deposition of iron-containing pigments in the tissues or, in the case of a deposition of iron in the organism, they cause the iron to be excreted, for example in haemochromatosis and haemosiderosis and also in cases of cirrhosis of the liver. They may also be used for excreting other metals, for example copper, from the organism.

Particularly valuable compounds are those of Formula I, in which R and R CO stand for acyl radicals which are independent of each other, .R represents also hydrogen or an unsubstituted or substituted hydrocarbon radical and among these compounds primarily those of Formula I in which R has the meaning given and R CO stands for acetyl or free or esterified succinyl, their salts with therapeutically acceptable acids or organic amines, alkalior alkaline earth metal hydroxides. Especially valuable compounds are the desferri-ferrioxamines B, C and D and their salts of the last-mentioned kind.

The trihydroxamic acids mentioned at the beginning, their salts and metal complexes may be obtained for example synthetically. Such synthesis consists in combining 3 mols of l-amino-S-hydroxylamino-pentane, 2 mols of succinic acid and one mol of a carboxylic acid or a compound which is convertible into one of these compounds, for example one that contains protecting groups or recatively converted functional groups, to form a compound of the Formula I, and, if desired, in a resulting compound having a terminal free amino group and a carboxyl group, condensing these groups intramolecularly to form a ring, and if desired, introducing or splitting off at any desired stage the acyl group or the hydrocarbon radical R and/or exchanging the radical R CO, and/ or forming or splitting off the O-acyl group and/or, if desired, esterifying any free carboxyl group in a resulting compound and/or forming the free compounds, the salts and/ or metal complexes of these compounds.

The starting materials mentioned may be condensed in succession, or individual radicals may be condensed together, and the resulting constituent parts then combined to form the entire chain or the ring.

Compounds convertible into l-amino-S-hydroxylaminopentane are, for example, pentanes of the formula wherein Z and Z represent an amino group and a radical convertible into a hydroxylamino group, respectively, or, conversely, a radical convertible into an amino group and a hydroxylamino group, respectively.

Radicals convertible into an amino or hydroxylamino group are, for example, reactive esterified hydroxyl groups, e.g. hydroxyl groups esterified with hydrohalic acids or sulfonic acids which on reaction with ammonia or hydroxylamino yield the amino or hydroxylamino group; furthermore, groups that can be converted into the amino or hydroxylamino group by reduction, e.g. the nitro group, the hydroxylimino group, or the nitrile group. Above all, the groups convertible into the amino or hydroxyl amino group comprise the amino or hydroxylamino groups protected by radicals that can be split off by hydrolysis or reduction. Such protective groups are, for example, acyl groups, especially carbobenzoxy, phthaloyl, trifluoracetyl groups, primarily the tertiary butyloxycarbonyl group, and also e.g. the tosyl and trityl radicals. These protective groups can be split off in known manner by treatment with hydrolyzing or hydrogenolyzing agents.

Compounds convertible into succinic acid or the second carboxylic acid are for example the halides, anhydrides, azides, imides, imidazolides or. esters of succinic acid or the second carboxylic acid.

Any carboxyl group not participating in the reaction is protected advantageously by esterification, e.g., with methanol, benzyl alcohol or para-nitro-benzyl alcohol.

When the condenation involves the free carboxyl groups, it is preferable to use a condensing agent, e.g. a carbodiimide, such as dicyclohexylcarbodiimide.

The synthesis of the trihydroxamic acids is advantageously performed according to this diagram of formulae:

RCO stands for an acyl group.

Like the compound Ib, the product mentioned under IIIa can also be converted in stages into the dihydroxamic acid and then into the trihydroxamic acid IV.

In this novel process, it has been found to be of particular advantage to form the N-substituted tetrahydro-3,6 dioxo-1,2-oxazines mentioned under II. These latter can be obtained from the compounds mentioned under Ie by treatment with a condensing agent, e.g. a carbodiimide, such as dicyclohexyl-carbodiimide. By this treatment, in a single process step, the terminal carboxyl group of the compounds mentioned under 1e is converted to form a 19 reactive group, and the N-hydroxyl group is protected.

The trihydroxamic acids mentioned at the beginning or their salts may also be obtained when a metal complex of a compound of the Formula I, in which R and CO-R have the meanings given above, especially an iron complex, is treated with a mineral acid, a strong alkali or a complex forming substance, e.g. 8-hydroxy-quinoline, and the resulting metal-free compound is isolated and, if desired, at any stage of the procedure the acyl group or the hydrocarbon radical R is introduced or split off and/ or the radical COR2 is exchanged and/ or an O-acyl group is formed or split off and/ or, if desired, in a resulting compound having a free carboxyl group, such group is esterified and/or the free compound or a salt is formed.

In reacting the metal complexes mentioned with mineral acids, especially hydrochloric acid, it is preferable to extract first the metal, e.g. iron, from the aqueous acid solution by means of a suitable solvent, for example, ether, then to render the solution nearly neutral, and then to extract the metal-free compound with a solvent, e.g. n-butanol.

When a solution containing the metal complex is treated with a strong alkali, the metal usually separates as the hydroxide in the form of fine flakes and can be isolated, for example, by filtration or centrifuging. After that, upon neutralization or slight acidification of the solution, the metal-free compound can be extracted as described above.

When a complex forming substance is used to remove the metal, for example 8-hydroxy-quinoline, the reaction is preferably performed in a lower alkanol, such as methanol. The precipitated metal complex is separated and any excess precipitant, such as 8-hydroxy-quinoline extracted from the aqueous solution, e.g. with chloroform, for example, after concentrating the filtrate.

In the resulting trihydroxamic acid any free l-amino group present may be acylated, for example, with an acid anhydride in a buffered alcoholic solution or with an acid halide in an aqueous, weakly alkaline medium. Any acyl derivatives formed can be converted into the O-unsubstituted N -acyl compounds e.g. by means of ammonia. The substitution of the l-amino group by a hydrocarbon radical, e.g. the m-dinitrophenyl radical, or the exchange of the radical CO-R can be performed in per se conventional manner.

N-acyl radicals that can be split off hydrogenolytically, for example the carbobenzoxy radical, can be split off in known manner at any stage. A free carboxyl group in a resulting compound, e.g. in desferri-ferrioxamine G, can be esterified by a method known in peptide chemistry.

There can be used as starting materials natural ferrioxamines and derivatives obtainable therefrom, for example, the N R compounds obtainable from the ferrioxamine B or G, in which R has the meaning given above, or the derivatives of ferrioxamine G or its N -R compounds having an esterified terminal carboxyl group, or the compounds obtained by total synthesis. The substitution of the l-amino group of ferrioxamine B or G is performed in the usual manner. The N -acyl compounds are obtained, for example, by reacting said ferrioxamines with an acid anhydride in alcoholic solution. The free terminal carboxyl group in ferrioxamine G or its N -acyl derivatives can be esterified by the usual methods. The esterification is advantageously performed with diazo compounds.

Depending on the procedure followed, the compounds are obtained in the free form or in the form of their salts. From the salts the free compounds can be obtained in the per se conventional manner. Likewise, the free compounds can be converted into the aforementioned acid addition salts or, if desired, into the alkali metal or alkaline earth metal salts or the salts of organic bases.

The O-unsubstituted compounds are converted into the metal complex by reacting them with a corresponding metal salt, for example the salt of a mineral acid, such as a metal chloride, sulfate or nitrate, or the salt of an organic acid, such as a metal acetate or metal sulfonate, or by reacting them wtih a metal alcoholate, for example, a metal ethylate.

The invention relates also to pharmaceutical preparations containing desferrioxamines or derivatives of the latter as above described.

Such pharmaceutical preparations therefore contain trihydroxamic acids of Formula I in which R and R have the meaning indicated above.

The preparations are capable of excreting iron from the organism by binding it in the form of an iron complex compound which is called ferrioxamine. Thus, in the case of deposition of ferriferous pigments in the organism they bring about excretion of the iron, for example in haemochromatosis and haemosiderosis and in cirrhosis of the liver. These properties are demonstrated for desferrioxamine B as follows:

Iron excretion in the dog Normal dogs were given a single intravenous or subcutaneous injection of 50 mg. desferrioxamine B per kg. body-weight in the form of a 10% solution. The excretion of ferrioxamine and iron in the urine was then observed for 4 days. It was found that the highest concentrations of ferrioxaminei.e. of the amount excreted within 72 hoursappeared in the urine within the first 24 hours after administration of desferrioxamine. During this period 2326% of the desferrioxamine B dose could be traced in the urine. 13l4% of it was the form of ferrioxamine, which corresponds to approx. 2 mg. iron. Following a daily subcutaneous dose of 50 mg desferrioxamine B per kg. body-weight for 6 days in dogs, ferrioxamine first appeared in the urine after only 24 hours and the urinary levels of this compound then remained practically constant throughout the duration of the test. Altogether 1819% of the desferrioxamine B administered was traced in the urine during an observation period of 8 days; 16-18% of it was in the form of ferrioxamine, which corresponds to approx. 10 mg. iron.

Other pharmacological effects In rabbits anaestetized with urethane, doses as high as 30 mg. desferrioxamine B per kg. are required to produce a temporary, slight fall in blood pressure; which is to be regarded as unspecific. No effect on respiration can be demonstrated.

Clinical effect Preliminary clinical studies on normal test subjects and on patients with haemochromatosis and haemosiderosis have shown that the intravenous or intramuscular administration of 400800 mg. desferrioxamine B in divided doses spread over the day leads to a considerable excretion of iron in the urine, the color of which may turn brownish red. The iron excretion is preceded by a rise in the plasma iron levels. In normal tests subjects the iron excretion amounted to some 10-20 mg. per day initially and decreased slightly later on; desferrioxamine has not yet been administered to normal test subjects on a long-term basis. The amounts of iron excreted by normal test subjects are of the same order of magnitude as the values observed in dogs, due allowance being made for the difference in body-weight. In patients with haemochromatosis, iron excretion was 10-20 mg. daily at the beginning of treatment. Where the preparation was administered daily for several weeks, these patients excreted iron in quantities ranging from 20 to 80 mg. daily. There was also evidence that liver function, concomitant diabetes, and heart failure due to the haemochromatosis were improved at the same time. Pigmentation of the skin decreased appreciably in some patients in the course of treatment lasting 2-3 weeks.

Desferrioxamine B has a low acute toxicity. In the mouse, the mean lethal dose (LD by the intravenous route is 340 mg./kg., and by the subcutaneous route 1,600

mg./kg. No signs of toxicity have been observed following oral administration of as much as 3 g./ kg. The intoxication picture is not specific. In rats, the mean lethal dose (LD by the intravenous route is 520 mg./kg., overt signs of intoxicationin the form of paralysis and spasm-occurring only in response to sublethal doses. If the preparation is administered subcutaneously or orally, the animals can survive a single dose of 1 g./kg. without showing signs of intoxication. The preparation is well tolerated locally in the rabbit eye in a solution of up to 3%.

The use of desferrioxamine B may be considered in the treatment of all diseases involving pathological deposition of iron in the organism, particularly haemochromatosis and haemosiderosis of varying aetiology, which may be associated, for example, with haemolytic anaemia and achrestie anaemia, or may follow frequent blood transfusions, or in the treatment of liver cirrhosis, a disease in which an elevated iron concentration can frequently be demonstrated in the liver.

The daily doses employed hitherto in human beings have ranged from 400 to 1200 mg. i.v. or i.m. Intravenous administration should be made as a drip infusions over a period of 610 hours. The contents of the ampoules should be dissolved or diluted in physiological saline prior to intravenous injection or infusion.

Especially valuable are preparations which contain compounds of Formula I in which R has the meaning given above and R CO stands for the acetyl radical or the free or esterified succinyl radical, their salts with therapeutically useful acids or organic amines, alkali or alkaline earth metal hydroxides. Especially preferred are the above desferri-ferrioxamines B, G, D and E and their salts of the last-mentioned type.

The aforementioned trihydroxamic acids which are the active principle of the new preparations may be obtained by treating iron complexes of compounds of Formula I in which R and COR have the meanings given, with mineral acids, strong alkalies or complex-forming substances, for example 8-hydroxy-quinoline or N-nitrosophenylhydroxylamine amonium salt, isolating the resulting iron-free compounds and, if desired, introducing into resulting compounds at any stage the acyl group or the hydrocarbon radical R or, if desired, splitting it off and/or exchanging the radical CO-R and/or forming O-acyl groups or eliminating them and/or, if desired, in resulting compounds with a free carboxyl group esterifying the latter, and/ or forming the free compounds or the salts.

The new pharmaceutical preparations contain the above defined trihydroxamic acids in admixture with an organic or inorganic pharmaceutical excipient suitable for enteral or parenteral administration. They are prepared from the starting materials by conventional methods. Suitable excipients are substances that do not react with the new compounds, such, for example, as gelatine, latcose, glucose, sodium chloride, starches, magnesium stearate, talc, vegetable oils, benzyl alcohols, gums, polyalkylene glycols, white petroleum jelly, cholesterol or other known medicinal excipients.

The pharmaceutical preparations may be in solid form, for example, capsules, dragees, powders, suppositories, vials or in liquid form, as solutions, suspensions or emulsions. They may be sterilized and/ or contain assistants such as preserving, stabilizing, wetting or emulsifying agents. They may also contain further therapeutically useful substances. The new compounds may also be used in veterinary medicine, for example in one of the forms mentioned above.

The following examples illustrate the invention.

EXAMPLE 1 Streptomyces pilosus strain NRRL 2857 is grown submerged on a nutrient solution containing per liter of tap water 20 grams of soybean flour and 20 grams of mannitol. The nutrient solution is sterilized in the inoculation flasks or in the fermenters for 20-30 minutes under a pressure of 1 atmosphere gauge. The pH-value of the sterilized nutrient solution is 7.2-7.6. For the inoculation up to 20% of a partially sporulating vegetative culture of the aforementioned organism is used. Incubation is carried out with vigorous shaking or stirring at 24-30 C., cultures in fermenters being aerated with about 2 volumes of air per volume of solution per minute. After an incubation period of 48-240 hours the culture solution displays its maximum content of ferrioxamines. The cultivation is discontinued, 0.1% of ferric chloride is added, and the mycelium together with other solid constituents is separated from the solution containing the bulk of ferrioxamines by filtration or centrifugation; if desired about 1% of a filter aid, for example Hyflo Supercel, is added to the culture solution prior to filtration. The filter residues are washed with water or aqueous methanol and the washings are combined with the culture filtrate. While stirring it continuously the resulting culture filtrate is treated with 2% of alumina, for example Frankonit. The mixture is thoroughly mixed and filtered and the resulting filtrate is subjected one or twice more to the adsorption operation. The filter residues are combined and washed repeatedly with Water and aqueous methanol and then eluted twice or three times with a 1:4-mixture of pyridine and water. The eluate is clarified by filtration and then concentrated in vacuo The resulting concentrates can be further worked up as they are (see Example 3), or a mixture of ferrioxamines in crude form an be isolated therefrom by means of freeze-drying.

When the nutrient solution described above is replaced by one which contains per liter of tap Water the following ingredients, and cultivation and working up are carried out in a similar manner concentrates of a similarly high ferrioxamine content are obtained:

Saccharose Sodium citrate Secondary potassium phosphate Magnesium sulfate 0.8 Copper sulfate mg 0.01 Manganese chloride mg 0.07 Ferric citrate -mg 20 Crude glucose 10 Soybean flour 10 Corn steep liquor 20 Sodium chloride 5 Sodium nitrate 1 Lime 10 tracer muqm Rape extraction shucks 20 Crude glucose 10 Secondary potassium phosphate 0.2 Lime 10 Flax meal 40 Crude glucose 10 Secondary potassium phosphate 0.2 Lime 10 23 Strain No. Streptomyces species 9578 S. grz'seoflavus (Krainsky) Waksman et Henrici.

15311 S. griseoflavus.

1.1686 S. pilosus Ettlinger et al.

23258 S. pilosus.

23305 S. pilosus.

17635 S. viridochromogenes (Krainsky) Waksman et Henrici.

18055 S. viridochromogenes.

6445 S. olivaceus (Waksman) Waksman et Henrici.

7346 S. olivaceus.

7437 S. olivaceus.

22083 S. aureofaciens Duggar.

22765 S. aureofacz'ens.

18822 S. galz'lseus Ettlinger et al.

14677 S. lavena'ulas (Waksman et Curtis) Waksman et Henrici.

21510 S. Iavendulae.

21837 S. polychromogenes Hagemann et al.

23217 S. polychromogenes.

23310 S. polychromogenes.

10112 S. griseus Waksman et Henrici.

13495 S. griseus.

7419 S. griseus.

EXAMPLE 2 Strain A 23978 of the species Streptomyces aureofaciens (Institute for Special Botany, Eidgenossische Technische Hochschule, Zurich) is grown as a submerged culture on a nutrient solution containing per liter of tap water. 20 grams of malt extract and 20 grams of distillers solubles. Cultivation and working up according to Example 1 yields culture filtrates having a similarly high content of ferrioxamines.

EXAMPLE 3 A culture of 60 liters is prepared and worked up as described in Example 1. The eluate (about 6 liters) obtained with a mixture of pyridine and water is concentrated in vacuo to 3 liters. 870 grams of ammonium sulfate are dissolved in this concentrate and the solution is clarified by filtration or centrifugation, if necessary with the addition of 1% of Hyflo Supercel. By shaking the solution 3 to 4 times with benzyl alcohol or isopropanol, the ferrioxamines are transferred into the organic solvent. The organic phases are combined and dried with the aid of sodium sulfate. An excess of ether or ethyl acetate is added and the precipitated ferrioxamines are filtered oiT.

Addition of a filter aid, for example Hyflo Supercel, prior to the precipitation facilitates the isolation of the precipiate, from which the ferrioxamines can be washed out with methanol or water. Those eluates are evaporated or lyophilized to yield a preparation of ferrioxamines in enriched form.

EXAMPLE 4 20 grams of sodium chloride per liter are added to a culture filtrate obtained according to Example 1 or 2. The clear solution is extracted three times with 0.1 volume of a mixture containing 1 part by weight of phenol in 1 part by volume of chloroform. The organic phases are combined, filtered in the presence of Hyflo Supercel, and treated with an excess of ether. When the solution is shaken repeatedly with a small amount of water, the ferrioxamines are transferred into the aqueous phases which are then combined and shaken twice with ether to remove the phenol completely. By freeze-drying an orange to brownish red preparation of ferrioxamines is obtained which can be separated by paper chromatography.

24 EXAMPLE 5 20 liters of culture solution are treated with 400 grams of Hyflo Supercel and 200 cc. of an aqueous ferric sulfate solution of 10% strength, and filtered. After adding 3.6 kg. of sodium chloride, the filtrate is extracted in a counter current extractor with 2 liters of a mixture of phenol and chloroform (1 g.:1 cc.), the extract dried over sodium sulfate and then allowed to run in the course of one hour into a well stirred suspension of 20 grams of Hyflo Supercel in 2 liters of ether and 10 liters of petroleum ether. After filtering the powdery mixture of filter aid and precipitate, the filtrate is washed with about 2 liters of ether and then eluted 5 times with 600 cc. of methanol on each occasion. The combined eluates are gently evaporated to yield 10 grams of crude ferrioxamine in the form of a brown red powder.

EXAMPLE 6 4 grams of ferrioxamine are distributed in the system n-butanol-benzyl alcohol-water-saturated aqueous sodium chloride solution-N-hydrochloric acid (100:200z300z60: 6) over stages each of cc. of organic phase and 100 cc. of aqueous phase. Evaluation of the distribution is carried out by biological testing and by measuring the extinction at 425 m of each fourth unit 2 cc. of upper and lower phase is taken and mixed with 32 cc. of methanol, a homogeneous solution being obtained whose concentration is suitable for both tests (cf, FIG. 1). The distribution fractions, put together in 4 groups according to this evaluation, contain, as shown by paper chromatography, in addition to the chief product ferrioxamine B (in distribution fraction 1 11), other red-colored compounds having an antisideromycin activity and which are designated as ferrioxamines A, C, D D E and F.

The distribution fraction III is agitated with 3 liters of petroleum ether. The deep red-colored aqueous phase is washed with chloroform, treated with sodium chloride up to a concentration of 10% and extracted exhaustively with a mixture of phenol and chloroform (1 g.:1 cc.). The phenol-chloroform extract is washed several times with 0.01 N-hydrochloric acid containing 10% of sodium chloride and filtered through a small column of 20 grams of Celite. The ingredients are precipitated by the addition of 25 grams of Hyflo Supercel. 500 cc. of ether and 1 liter of petroleum ether with stirring at 0 C. The powdery mixture of filter aid and precipitate is washed well with ether and then eluted with a little methanol, From the methanol eluate there are obtained on gentle evaporation 982 mg. of crude ferrioxamine B in the form of a brown-red powder.

The distribution fractions II, IV and V are worked up in the same manner.

EXAMPLE 7 1 gram of a preparation of ferrioxamine B prepared as described in Example 6 is subjected to zone electrophoresis according to J. Porath (Biochem. et Biophys. Acta 22, p. 151 [1956]) in a vertical glass column of 1.5 meters height and 2.6 centimeters diameter equipped with a cooling jacket and filled with cellulose powder. Electrolyte solution: 1/3 N-acetic acid, The substance is dissolved in 20 cc. of water and the deep red solution is poured over the column from the top end of the anode. At a voltage of 1600 volts and a current intensity of 30 milliamperes the orange red ferrioxamine zone (about 10 cm. high) migrates at a rate of 3.9 cm. per hour towards the cathode at the bottom end of the column. To increase the separating action of the column this 

